title: Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs).
abstract: Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS.
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, 2000 Rosario, Santa Fe, Argentina.
authors.forename: Mariana
authors.initials: M
authors.lastname: Martín
authors.forename: Nahuel Z
authors.initials: NZ
authors.lastname: Wayllace
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Valdez
authors.forename: Diego F
authors.initials: DF
authors.lastname: Gomez-Casati
authors.forename: María V
authors.initials: MV
authors.lastname: Busi
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D006003:Glycogen / Q000096:biosynthesis; D006006:Glycogen Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D060847:Metabolic Engineering; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D006003:Glycogen; D006006:Glycogen Synthase
keywords: CBM; Glycogen synthase; Starch binding domains; Starch synthase